THE NEPHROPATHIC ACTION OF VARIOUS DICAR- 
BOXYLIC ACIDS. THE FATE OF CERTAIN 
DERIVATIVES OF GLUTARIC AND ADIPIC 
ACIDS IN THE ANIMAL ORGANISM 


BY 


Revie eONNER GORLEY: 


A.B., University of Illinois, 1921 
A.M., University of Illinois, 1922 


THE LIBRARY OF THE 


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SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS 
POs eOranEt Or DOCTOR OF PHILOSOPHY IN;:CHEMISTRY 
IN THE GRADUATE SCHOOL OF THE UNIVERSITY 
OF ILLINOIS; 1924 


Reprinted from The Journal of Pharmacology and Experimental Therapeutics, 
Vol. XXVII, No. 2, March, 1926 


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Reprinted from Tap YOurnat or PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 
> Vol. X XVII, No. 2, March, 1926 
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THE NEPHROPATHIC ACTION OF THE DICARBOXYLIC 
ACIDS AND THEIR DERIVATIVES 


V. ALKYL-, HYDROXY-, AND KETO-ACIDS! 


RALPH C. CORLEY? wita WILLIAM C. ROSE 
From the Laboratory of Physiological Chemistry, University of Illinois, Urbana 


Received for publication August 28, 1925 
INTRODUCTION 


In the preceding papers of this series (11), (12), (13), (14) are 
reported the results of nephrotoxicity studies involving the 
injection of seven of the homologues of oxalic acid, and four of 
the hydroxy derivatives of these homologues. Of the acids 
tested, tartaric, mucic, and glutaric acids manifested marked 
effects upon the kidneys leading to considerable destruction of 
the tubules. ‘The other acids examined either failed to affect 
renal function, or, at most, led to a temporary inhibition in 
excretion followed by prompt recovery. The results suggested 
the desirability, (a) of testing the renal affects of several isomers 
of glutaric acid, and (6) of determining the influence upon renal 
toxicity of modifying the structure of glutaric acid by introducing 
an hydroxy or a ketonic group into the alpha position: Inas- 
much as tartaric acid is violently toxic, dl-malic acid very slightly 
toxic, and succinic acid non-nephrotoxic, it seemed possible, as 
suggested in a previous paper (11), that hydroxylation might 
lead invariably to an increase in the renal action of a dicarboxylic 
acid. This conception seemed to be rendered more likely by 


1 Aided by a grant from the Research Fund of the American Association for the 
Advancement of Science. 

2 The experimental data in this paper are taken from a thesis submitted by 
Ralph C. Corley in partial fulfillment of the requirements for the degree of Doctor 
of Philosophy in Physiological Chemistry in the Graduate School of the Univer- 


sity of Illinois. 
165 


THE JOUR. OF PHARM. AND EXPER. THERAP., VOL, XXVII, NO, 2 


2 527930 


166 RALPH C. CORLEY WITH WILLIAM C. ROSE 


the observation that mucic acid is far more toxic than adipic acid 
(14). If this effect always occurs, a-hydroxyglutaric acid should 
be more destructive to the kidneys than glutaric acid itself. 
On the other hand, if nephrotoxicity depends upon the inability 
of the organism to destroy the substance in question, the intro- 
duction of an hydroxy or a ketonic group into the alpha position 
of glutaric acid might diminish the renal effects of the latter 
through enabling the organism to transform the substituted 
glutaric acid into the non-nephrotoxic succinic acid. 

With these considerations in mind we have (a) determined the 
influence upon the kidneys of three isomers of glutaric acid, 
namely, dimethyl malonic, ethyl malonic, and pyrotartaric acids. 
For purposes of comparison we have tested also the action of 
methyl malonic acid as an illustration of an isomer of the non- 
toxic succinic acid. (6) Similar experiments have been made 
with the a-hydroxy and o-ketonic derivatives of glutaric and 
adipic acids. It seemed not improbable that oxidation of the 
derivatives of adipic acid might lead to the production of glu- 
taric acid in sufficient quantities to exert detrimental effects 
upon renal function. (c) For reasons which will be set forth 
later, some of the acids were administered to completely diabetic 
animals with a view to obtaining further light upon their fate in 
the body. 


EXPERIMENTAL 
Methods 


Normal rabbits served as the experimental animals through- 
out the nephrotoxicity experiments. Previous to the administra- 
tion of the acids the animals were permitted to fast for twenty- 
four to thirty-six hours. One or more blood samples were 
then removed from each animal, and subjected to analysis. 
The acids were injected subcutaneously as their sodium salts, the 
injection solutions always being exactly at the neutral point. 
Blood samples were withdrawn at the intervals indicated in the 
tables. Kidney function was also tested by the Geraghty- 
Rowntree method (3). In order to exclude dietary influences (15) 


167 


ALKYL-, HYDROXY-, AND KETO-ACIDS 


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168 RALPH C. CORLEY WITH WILLIAM C. ROSE 


the animals were permitted to fast throughout the experiments, 

care being taken to avoid prolongation of the fasting periods to 

the point where blood nitrogen values are increased. The acids 

employed were either synthetic products prepared in this labora- 

tory, or commercial preparations which were thoroughly purified 

by repeated recrystallizations. Their origin and melting points 
TABLE 2 


Methyl malonic acid 
Rabbit 46, male, 1800 grams 


BLOOD 


Spal ea 
Z, Zz fs 5 A g 
DATE Z a a ° 2 = NOTES, ETC, 
ele cea cies 
PO apse |e 
grams el mgm. | mgm Rati 
January 28.2255 No food; water ad lib. 
January 29.0%... .$ 70* | 44.0) 1.5 | 0.47) 8:00 a.m., 7 ec. of blood. 1:15 
p.m., dye test 
PADUET VIGO nace 41.5| 1.5 | 0.48} 8:00 a.m., 7 ce. of blood 
January 30....... 2.0 | 45f 9:30 a.m., acid injected as 
sodium salt in 17 cc. of water. 
3:16 p.m., dye test 
PRIMATOL gers <a 54.7| 1.6 | 0.45} 8:00 a.m., 7 ec. of blood 
February 1....... 49 .0| 1.6 | 0.46) 8:00 a.m., 7 cc. of blood 
February 2....... 69t | 43.0) 1.6 | 0.45) 8:00 a.m., 7 cc. of blood. 9:18 


a.m., dye test. Discontinued 


* First period, 3 cc. of urine containing 44 per cent dye; second period, 3 cc. 
of urine containing 26 percent dye. Total 70 percent. 

} First period, 3 cc. of urine containing 20 per cent dye; second period, 3 cc. 
of urine containing 25 per cent dye. Total 45 per cent. } 

t First period, 3 cc. of urine containing 51 per cent dye; second period, 4 cc. 
of urine containing 18 percent dye. Total 69 per cent. 


are shown in table 1. For the methods of analysis, and other 
details regarding technique, the reader is referred to a previous 
communication (11). 


The effect of tsomers of glutaric acid upon renal function 


Three experiments were made with each acid, but inasmuch 
as the results in all cases were perfectly consistent, we are present- 


ALKYL-, HYDROXY-, AND KETO-ACIDS 169 


ing the data obtained in only one experiment with each substance. 
In table 2 are shown the results of a single experiment with methyl 
malonic acid. In tables 3 to 5 inclusive are presented data 
secured with the isomers of glutaric acid. In no case is there 
distinct evidence of marked renal toxicity. The most pro- 


TABLE 3 
Dimethyl malonic acid 
Rabbit 47, male, 1760 grams 


BLOOD 


a 
fa] 
a 
2 a | 4 
DATE g é eB 3§ a NOTES, ETC, 
Bes aoeh SEA lg ae 
Bal Ba | Se mec ks 
= a Zi (oo) Zi 
erams send Gees Ege ae 
October 20....... No food; water ad lib. 
October 21... .... 78* | 43.0} 1.1 | 0.54} 9:00 a.m., 6 ce. of blood. 10:30 
a.m., dye test 
October 2222.03... 44.0) 1.1 | 0.55) 9:40 a.m., 6 cc. of blood 
October 22... ..%. 2.0 \\61T 10:45 a.m., acid injected as 


sodium salt in 15 ec. of water. 
8:50 p.m., dye test 


Octoberi2aneay 3% 49.1; 1.1 | 0.51} 8:45 a.m., 6 cc. of blood 

October'242:5; %: x. 59t | 45.8) 1.3 | 0.50} 9:45 a.m., 7 cc. of blood. 7:53 
p.m., dye test 

October 25....... 43.1} 1.3 | 0.51] 9:25 a.m., 6 ec. of blood 

October 26....... 44.5) 1.1 | 0.52) 9:50 a.m., 5 ec. of blood. Dis- 


continued 


* First period, 10 cc. urine containing 61 per cent dye; second period 8 cc. of 
urine containing 17 per cent dye. Total 78 per cent. 

| First period, 6 cc. urine containing 50 per cent dye; second period, 7 cc. urine 
containing 11 percent dye. Total 61 per cent. 

t First period, 10 cc. urine containing 46 per cent dye; second period, 10 ce. 
urine containing 13 percent dye. Total 59 per cent. 


nounced effect was observed in the case of ethyl malonic acid 
(table 4). Following the administration of 2 grams of this sub- 
stance the non-protein nitrogen almost doubled, accompanied 
by a slight increase in creatinine. ‘These effects, however, were of 
short duration, and were followed by a rapid return to normal. 
In all of the experiments, more or less inhibition in phenol- 


170 RALPH C. CORLEY WITH WILLIAM C. ROSE 


sulphonephthalein excretion was observed soon after the adminis- 
tration of the acid. But this effect also was quite temporary in 
nature, in every animal the dye excretion returning to normal 
before the end of the experiment. In no case was the toxicity 
of the acid comparable to that produced by glutarie acid. 


TABLE 4 
Ethyl malonic acid 
Rabbit 48, male, 2200 grams 


BLOOD 


: 
fl 
zZ alg 
DATE c q a g gol ne NOTES, ETC, © 
A oil) ed Cente 
i esc eck bed 
< a Ai 6) ZG 
grams pice’ mgm, | mgm “sha 
January see tals ; No food; water ad lib. 
January COM As 77* | 46.1) 1.6 | 0.50} 8:30 a.m., 7 cc. of blood. 10:48 
a.m., dye test 
JANUATYsLO ete se sc: 38.8} 1.6 | 0.50} 8:30 a.m., 7 cc. of blood 
Januarye100. 4 lao 2.0 | 43f 11:20 a.m., acid injected as 


sodium salt in 15 cc. of water. 
7:18 p.m., dye test 


Januaryel linc. eise 64.3) 1.8 | 0.49) 8:30 a.m., 7 ec. of blood 
January lee dons 25 52.9} 1.6 | 0.50} 8:30 a.m., 7 ce. of blood 
JROQUBTY vl sa 

January 1498783. 82f | 45.9) 1.8 | 0.50) 8:30 a.m., 7 ce. of blood. 9:50 


a.m., dye test. Discontinued 


* First period, 12 ce. of urine containing 60 per cent dye: second period, 7 cc. 
of urine containing 17 per cent dye. Total 77 per cent. . 

} First period, 6 cc. of urine containing 22 per cent dye; second period, 5 cc. of 
urine containing 21 per cent dye. Total 48 per cent. 

t First period, 5 cc. of urine containing 63 per cent dye; second period, 2 ce. of 
urine containing 19 percent dye. Total 82 per cent. 


It is evident from the above data that the presence of five 
carbons in a dicarboxylic acid does not necessarily indicate that 
the acid is capable of exerting detrimental renal effects. Kay 
and Raper (5) have recently shown that the presence of an a- 
methyl group does not hinder the normal course of oxidation of 
certain fatty acids. It is possible that the same principle may 
apply to dicarboxylic acids. In this case, the acids we have em- 


ALKYL-, HYDROXY-, AND KETO-ACIDS AL 


ployed would undergo oxidation along lines similar to those fol- 
lowed by malonic and succinic acids. It will be recalled that 
neither of the latter exerts destructive effects upon the kidneys 
eTpert 1D.) 
TABLE 5 
Pyrotartaric acid 
Rabbit 49, male, 1800 grams 


D 


BLOOD 


& 
5 
: a |e 
DATE | ae | 8a me NOTES, ETC. 
145 | 8). a 
41.38 | #8) 3 = 
Ae hip |) Sane ts 
AE ANS IS 2 ened 
srama| 2, | mom.| mom.| 24, 
Janitgny la: sree. No food: water ad lib. 
Janueryelor we. ; 83* | 47.4) 1.8 | 0.50) 8:10 a.m., 7 cc. of blood. 1:12 
p.m., dye test 
‘TOPE E aio Ue, 46.1} 1.8 | 0.51} 8:10 a.m., 7 cc. of blood 
ANUAPY: LOU casks es 2.0 | 88f 10:25 a.m., acid injected as 
sodium salt in 15 ce. of water. 
7:06 p.m., dye test 
JANUATY, Lace ques 47.1) 1.8 | 0.50} 8:10 a.m., 7 cc. of blood 
JEOUSEY 1Oi ease 49.5} 1.7 | 0.53) 8:10 a.m., 7 ec. of blood 
Panvaryesde ca 74t | 50.0} 1.7 | 0.51} 8:10 a.m., 7 cc. of blood. 9:03 


a.m., dye test. Discontinued 


* First period, 5 cc. of urine containing 71 per cent dye; second period, 4 cc. of 
urine containing 12 per cent dye. Total 83 per cent. 

{ First period, 4 cc. of urine containing 20 per cent dye; second period, 3 cc. of 
urine containing 18 percent dye. ‘Total 38 per cent. 

{ First period, 3 cc. of urine containing 63 per cent dye; second period, 5 cc. of 
urine containing 11 percent dye. ‘Total 74 per cent. 


The effect of a-hydroxy- and a-ketonic-derivatives of glutaric and 
adipic acids upon renal function 


In tables 6 and 7 are presented the results of two typical experi- 
ments with a-hydroxy- and a-ketoglutaric acids respectively. 
In neither of these experiments did the administration of 2 grams 
of the acid in question produce more than a very slight nitrogen 
retention, or temporary slowing in the rate of dye elimination. 
Indeed, in the case of the ketonic acid no effects whatsoever are 
to be observed upon renal function. These results are in striking 


172 RALPH C. CORLEY WITH WILLIAM C. ROSE 


contrast to the pronounced nephrotoxicity of unsubstituted 
glutaric acid. It would appear that thé introduction of the hy- 
droxy and ketonic groups renders the resulting compounds less 
stable in the animal organism, and provides a means whereby 
they can be converted through oxidation into the non-toxic 
succinic acid. Whether this conception is correct or not, it is 
undoubtedly true that the alterations in structure destroy practi- 


TABLE 6 
Alpha-hydroxyglutaric acid 
Rabbit 50, male, 1500 grams 


BLOOD 


a 
= 
+ 
z a | ¢ 
DATE g Ae | 88 2 NOTES, ETC. 
a chat te ge a= 
= Aen Wey SA NL (tte = 
a |ee]| se) 8 | & 
Dut Cammy ec Oe) 
srame| 22, | mom.| mom.| 22 
March 19 5 ue aon No food; water ad lib. 
Ware 20s. einem 76* | 46.2)} 1.6 | 0.51} 8:00 a.m., 7 ec. of blood. 7:10 
p.m., dye test 
Marcne2li nie akc ae 48.2) 1.6 |. 0.52) 8:00 a.m., 7 cc. of blood 
Merch 21 eich ein 2.0 | 41T 2:00 p.m., acid injected as 
sodium salt in 13.5 ec. of 
water. 7:25 p.m., dye test 
Marchi22uneor in 71.2) 1.9 | 0.56} 8:00 a.m., 7 cc. of blood 
March 23 ona. « 59.6] 1.6 | 0.55} 8:00 a.m., 7 cc. of blood 
March 24. 2022... Died during night 


* First period, 5 cc. of urine containing 64 per cent dye; second period, 4 cc. of 
urine containing 12 per cent dye. Total.76 per cent. 

} First period, 3 cc. of urine containing 22 per cent dye; second period, 2 ce. of 
urine containing 19 per cent dye. Total 41 per cent. 


cally all of the detrimental properties inherent in unsubstituted 
glutaric acid. These data appear to substantiate the correctness 
of the suggestion made in a former communication (11), namely, 
that not chemical structure per se, but the ability or inability of 
the animal cells to accomplish the oxdation of the compound, is 
the important point in determining whether a given substance is 
or is not nephrotoxic. We shall return to this aspect of the 
question later in this communication. 


ALKYL-, HYDROXY-, AND KETO-ACIDS 173 


Inasmuch as modification of the alpha position in glutaric 
acid is accompanied by such marked effects upon physiological ac- 
tion, it seemed not unreasonable to assume that a similar substi- 
tution in the alpha position of adipic acid might be associated 
with an increase in nephrotoxicity, through rendering the result- 


TABLE 7 
Alpha-ketoglutaric acid 
Rabbit 51, male, 2050 grams 


BLOOD 


a 
DATE = ai 2 5 a NOTES, ETO, 
Be ls Sil ore Naar Bos 
2 “B= 6) 
grams nae mgm, | mgm a 
PBNUAT VS ce ce No food; water ad lib. 
JONUAr ye Fae ae. 79* | 51.1} 1.7 | 0.48} 8:15 a.m., 7 cc. of blood. 10:34 
a.m., dye test 

January (Ulery aa 50.8] 1.8 | 0.50) 8:15 a.m., 7 ec. of blood 
JanuaryalOxe ie. 2.01774 11:50 a.m., acid injected as 


sodium salt in 14.5 cc. of 
water. 7:13 p.m., dye test 


AINULALY: Ulla. aaeir 55.6] 1.6 | 0.41] 8:10 a.m., 7 ec. of blood 
Paniaryo lea on, we 48.9} 1.5 | 0.43} 8:15 a.m., 7 cc. of blood 
Janiarvalsa eros 

January 149023 ,)... 84t | 50.0) 1.6 | 0.46) 8:10 a.m., 7 cc. of blood. 9:45 


a.m., dye test. Discontinued 


* First period, 15 cc. of urine containing 62 per cent dye; second period, 8 cc. 
of urine containing 17 percentdye. Total 79 percent. 

t+ First period, 4 cc. of urine containing 58 per cent dye; second period, 6 cc. of 
urine containing 19 percent dye. Total 77 percent. 

t First period, 25 cc. of urine containing 74 per cent dye; second period, 10 cc. 
of urine containing 10 per cent dye. Total 84 per cent. 


ing compounds capable of oxidative transformation into glutaric 
acid. Contrary to our expectations, however, the administration 
of a-hydroxy- and a-ketoadipic acids was not followed by more 
pronounced effects than was occasioned by the injection of the 
corresponding glutaric acid derivatives (cf. tables 8 and 9). 
These results would seem to indicate that the formation of glu- 
taric acid from the derivatives of adipic acid, if it occurs at all, 


174 RALPH C. CORLEY WITH WILLIAM C. ROSE 


is a very slow process which does not lead to an accumulation of 
sufficient quantities of the former to injure the kidneys. 

In order to obtain further light upon the fate of the substituted 
acids it seemed necessary to administer them to completely 
diabetic animals and thereby determine their influence upon 
sugar excretion. If our contention is correct concerning the 

TABLE 8 
Alpha-hydroxyadipic acid 
Rabbit 52, male, 1930 grams 


BLOOD 


ACID ADMINISTERED 


a | A 
DATE EEE: gs NOTES, ETC. 
qo.1 ke | 8 | _ 
y Z 6) Z 
grams ae mgm. | mgm bat 
April 13 tee ts No food; water ad lib. 
Aprillaiee ca: ee 73* | 43.3) 1.4 | 0.47] 9:00.a.m., 7 cc. of blood. 7:29 
p.m., dye test 
ATID Sale. eet 42.1} 1.3 | 0.46] 9:00 a.m., 7 cc. of blood 
ADTINLD Ort 2. shies 2.0 | 35f 12:35 ‘p.m. acid injected as 
| sodium salt in 12.5 cc. of 
water. 7:22 p.m., dye test 
Aprils Gees hee 40.4) 1.5 | 0.46) 9:00 a.m., 7 cc. of blood 
ADU eer ns 35 34.6) 1.5 | 0.45} 9:00 a.m., 7 cc. of blood 
Anrili1 9 eee sk 74t | 38.3) 1.5 | 0.46) 9:00 a.m., 7 cc. of blood. 3:35 


p.m., dye test. Discontinued 


* First period, 4 cc. of urine containing 50 per cent dye; second period, 4 cc. of 
urine containing 23 per cent dye. Total 73 per cent. 

{ First period, 4 cc. of urine containing 20 per cent dye; second period, 3 cc. 
of urine containing 15 per cent dye. Total 35 per cent. 

{ First period, 3 ce. of urine containing 51 per cent dye; second period, 6 cc. of 
urine containing 23 per cent dye. Total 74 per cent. 


derivatives of glutaric acid, that they are readily oxidized to 
succinic acid, one would expect the administration of either of 
these substances to the phlorhizinized animal to lead to a reap- 
pearance of three-fifths of the carbon of the acid in the form of 
extra glucose. This assumption is based upon the findings of 
Ringer and his associates (10) that succinic acid yields extra 
glucose in the diabetic dog, probably to the extent of three- 


ALKYL-, HYDROXY-, AND KETO-ACIDS 175 


fourths of the carbon present in the acid. On the other hand, if 
the derivatives of adipic acid are incapable of oxidative change, 
or are oxidized slowly to glutaric acid, one would not expect extra 
sugar excretion following their administration, since Baer and 
Blum (1), and Ringer (9) have shown that unsubstituted glutaric 
acid fails to lead to the production of extra glucose. With these 


TABLE 9 
Alpha-ketoadipic acid 
Rabbit 53, male, 2070 grams 


= , 
A & BLOOD 
e | 
B g 
DATE a 5 q NOTES, ETC, 
| & | Se! 2 
2 | 3 | Be] 3 
Amilece Baa lmeelacs 
<q Ay Z s G 
grams] 2, | mom.| mam. | 22 
iViarohiy lie 32.28. No food; water ad lib. 
IVIGECUR Zt, 82* | 38.5} 1.5 | 0.50) 10:00 a.m., 7 cc. of blood. 7:15 
p.m., dye test 
WEArGa IS cate ere: 41.6} 1.5 | 0.52] 10:00 a.m. 7 ce. of blood 
Ividrchsls. wa aan 2.0 | 507 11:55 a.m., acid injected as 
sodium salt in 12.5 ec. of 
water. 7:20 p.m., dye test 
Marchy14.9 4.22... 44.1| 1.6 | 0.50} 10:00 a.m., 7 ec. of blood 
Marchiel ntsc. cece 
Mariah: oot 47.2} 1.6 | 0.50} 10:00 a.m., 7 cc. of blood. Dis- 


continued 


* First period, 3 cc. of urine containing 74 per cent dye; second period, 4 cc. of 
urine containing 8 percent dye. Total 82 per cent. 

} First period, 2 cc. of urine containing 34 per cent dye; second period, 3 cc. of 
urine containing 16 percent dye. Total 50 percent. 


considerations in mind, experiments were made with both deriva- 
tives of glutaric acid, and with a-ketoadipic acid. 


The fate of a-hydroxy- and w-ketonic-derivatives of glutaric and 
adipic acids in the completely diabetic animal 


Dogs were used as the experimental animals. After prelimi- 
nary fasting periods of two to four days, the animals were ren- 


176 RALPH C. CORLEY WITH WILLIAM C. ROSE 


dered diabetic by the usual method of injecting phlorhizin sus- 
pended in olive oil (1 gram per 7 cc.). Under such conditions 
satisfactory G:N ratios were almost immediately established. 
The urines were collected in periods of twenty-four hours, pre- 
served with toluene, and kept on ice until the analyses were 
begun. At exactly the same hour each day the animals were 
catheterized, the bladders washed out with warm boric acid 
solution, and the phlorhizin then administered subcutaneously. 
The urines were analyzed for total nitrogen and sugar. Nitrogen 


TABLE 10 
Sugar formation from alpha-hydroxyglutaric acid 
Dog 4, 14.1 kgm. 


URINE 


DATE 


CALCULATED 


ACID ADMINISTERED 
NITROGEN 


BLOOD NON-PROTEIN 
EXTRA GLUCOSE 
EXTRA GLUCOSE 


grams ce, grams | grams mgm. | grams | grams 
March-p3tik. Ais $9 sot oe 1400 | 35.3 | 10.56} 3.35 
War eid ts Aeie o paeiee 1150 | 39.3 | 11.78} 3.34 | 32.7 
Marcin oie... wel pole nets 10 Of} 15007) 43.5 |10-56}94-.12 6.08 | 5.94 
Mearelr 26 Peta 8ak, bs. Sete 1070] 19.1 4.80} 3.98 | 35.5 


* Animal began fasting March 19. One and one-half grams of phlorhizin were 
given daily in olive oil beginning March 22. 

+ Acid injected subcutaneously as its sodium salt in three equal doses at noon, 
2:00 p.m., and 4:00 p.m. 

t Animal died during the night. Not acomplete day’s urine. 


determinations were made by the Kjeldahl-Gunning method, and 
sugar by the procedure of Shaffer and Hartmann (16). In order 
to exclude the possibility of nitrogen retention, which might 
invalidate our conclusions, determinations of non-protein nitro- 
gen of the blood were made the morning before and the morning 
after the administration of the acid to be tested. The blood 
samples were taken from an ear vein or from the jugular. 

Two or more concordant experiments were made with each of 
the three acids. The results of a single typical experiment with 


a; 


ALKYL-, HYDROXY-, AND KETO-ACIDS 177 


each compound are shown in tables 10 to 12 inclusive. In eal- 
culating the extra glucose excretion, we have subtracted from the 
G:N ratio of the day the acid was administered, the average of 
the two preceding days and of the following day (if the latter 
were available), and then multiplied the resulting figure by the 
nitrogen excretion of the experimental day. As will be observed 
from the data, both of the glutaric acid derivatives led to the 
excretion of extra glucose. In the case of the a-hydroxyglutaric 
acid, 5.94 grams of extra glucose were recovered as against the 


TABLE 11 
Sugar formation from alpha-ketoglutaric acid 
Dog 1, 10.4 kgm. 


URINE 


DATE 


CALCULATED 


ACID ADMINISTERED 
NITROGEN 


BLOOD NON-PROTEIN 
EXTRA GLUCOSE 
EXTRA GLUCOSE 


Q 
zi 
ume | cose | nitro- : 
grams cc, grams grams mam, grams grams 
HO@DrTUATY, 2224 Ga eee ae 1050 | 34.4 | 8.33 | 4.13 
Hebruary: Jone ere ara. 1340 | 27.6 | 8.28 | 3.34 
Bebrugry 240. aston ee 1650 | 26.0 | 8.16 | 3.19 | 32.6 
FRDFUSIY Zou. oo see 10.0t| 1300 | 28.5 | 6.86 | 4.16 6.16 | 5.10 
HG DTUHARY: 260 ps Soe hee 590 | 23.7 | 6.37 | 3.72 | 32.6 


* Animal began fasting February 18, and from that time received 1 gram of 
phlorhizin in olive oil every twelve hours. 

+ Acid injected subcutaneously as its sodium salt in three equal doses at 3:30 
p.m., 5:45 p.m., and 10:45 p.m. © 


calculated quantity of 6.08 grams, assuming that three-fifths of 
the carbon is transformed into glucose. With the a-ketoglutaric 
acid, 5.10 grams of extra glucose were recovered as against the 
calculated quantity of 6.16 grams. In another experiment, the 
details of which are omitted, the administration of 15 grams of 
a-ketoglutaric acid was followed by the excretion of 10.28 grams 
of extra glucose as compared with the calculated mount of 9.25 
grams. It appears, therefore, that both of the glutaric acid 
derivatives are oxidized in such a fashion as to yield three-fifths 


178 RALPH C. CORLEY WITH WILLIAM C. ROSE 


of their carbon in the form of extra glucose. This would seem 
to offer rather convincing proof of an intermediate formation of 
succinic acid. It also serves to strengthen our conception that 
inability to undergo oxidative destruction is the determining 
factor in the nephrotoxicity of glutaric acid, and probably of 
other toxic dicarboxylic acids. 

In contrast to the behavior of the glutaric acid derivatives, 
a-ketoadipic acid entirely failed to yield extra glucose. Following 


TABLE 12 
Sugar formation from alpha-ketoadipic acid 
Dog 6, 23 kgm. 


URINE 


DATE 


ACID ADMINISTERED 
CALCULATED 


BLOOD NON-PROTEIN 
EXTRA GLUCOSE 
EXTRA GLUCOSE 


fs 
ee : ; 
Vol- | Glu- | °0°4 B 
ume | cose | Mitra) GIN | 3& 2 
7290) Wp RNR SEN ie EN ESB 1310 | 63.7 | 16.74) 3.81 
Apri coy Cnt aee amr 1500 | 64.2 | 17.33) 3.70 
ADI ath tae oe ee ee 16380 | 67.4 | 17.54) 3.84 | 30.9 
Apr tua bona 10.07} 1850 | 69.6 | 18.80) 3.71 5.62 0 
ADTILS Saieerie,. osc, cares 1800 | 53.7 | 14.62) 3.67 | 29.4 
ApriliG eee of asus one ee 1220 | 52.2 | 14.46) 3.61 


* Animal began fasting March 28. One and one-half grams of phlorhizin were 
given daily in olive oil beginning March 31. 

| Acid injected subcutaneously as its sodium salt in three equal doses at 2:00 
p.m., 5:00 p.m., and 8:00 p.m. 


the administration of 10 grams of the acid, no increase in glucose 
excretion was observed. If, contrary to our expectations, three 
of the six carbons present had been transformed into sugar, an 
extra glucose excretion of 5.62 grams would have occurred. 
Whether the acid is entirely resistant to oxidation, or whether it 
is transformed slowly into glutaric acid which is excreted, and 
thus fails to accumulate in nephrotoxic quantities, remains to. 
be determined. 


= —— we 


~~ 


ALKYL-, HYDROXY-, AND KETO-ACIDS 179 


DISCUSSION 


Of the nineteen acids which have been examined for nephro- 
toxicity in this series of investigations only three have been 
found to exert pronounced detrimental action upon the kidneys, 
namely, tartaric, mucic, and glutaric acids. The wide dissimi- 
larity in structure of these three compounds clearly indicates 
that there is not a definite relationship between chemical con- 
stitution and renal toxicity. This fact is further indicated by 
the observation that the introduction of an hydroxy group may 
increase nephrotoxicity, as illustrated in the behavior of tartaric 
and malic acids; may decrease toxicity, as in the case of the forma- 
tion of a-hydroxyglutaric acid from glutaric acid; or may exert 
practically no influence as seen in the action of adipic acid and its 
a-hydroxy derivative. Probably, as is stated above, the real 
determinant is the effect produced by the structural change 
upon the physiological stability of the resulting compound. If 
hydroxylation pronouncedly increases the ease of oxidation of the 
substance, toxicity is diminished. With the reverse effect upon 
oxidizability, renal toxicity is increased. 

It must be admitted that other factors may, and probably do, 
enter into the question of nephrotoxicity, but as far as our in- 
vestigations are concerned, the most evident factor is the ability 
or inability of the organism to destroy by combustion the foreign 
substance administered. 


SUMMARY 


1. Experiments made with methyl malonic, dimethyl malonic, 
ethyl malonic, and pyrotartaric acids show that neither of these 
substances is a distinctly nephrotoxic agent. The administra- 
tion of each produces a temporary slowing in the rate of phenol- 
sulphonephthalein elimination, and may lead to a slight, tempo- 
rary nitrogen retention. Inasmuch as three of these acids are 
isomers of the very toxic glutaric acid, it is apparent that the 
number of carbon atoms present in a dicarboxylic acid has per 
se no relation to its nephrotoxicity. 

2. The introduction of an a-hydroxy or an a-ketonic group into 


180 RALPH C. CORLEY WITH WILLIAM C. ROSE 


glutaric acid almost completely destroys its nephrotoxic effects. 
This is attributed to greater physiological instability of the re- 
sulting compounds as evidenced by their transformation into 
extra glucose in the completely diabetic animal. 

3. The introduction of an a-hydroxy or an a-ketonic group into 
adipic acid does not influence toxicity. This is interpreted as 
indicating that the substituted adipic acids are very slowly 
transformed into glutaric acid, which is excreted without accu- 
mulating in sufficient amounts to injure the kidneys. In con- 
trast to the behavior of a-ketoglutaric acid, a-ketoadipic acid 
entirely fails to yield extra glucose in the completely diabetic 
dog. | 

REFERENCES 


(1) Bazr, J., AND Buu, L.: Beitr. chem. Physiol. u. Pathol., 1907, x, 80. 
(2) Gautt, H.: Compt. rend. Acad., 1909, exlviii, 1113. 
(3). Gpracuty, J. Y., AND Rowntree, L. G.: Jour. Amer. Med. Assoc., 1911, 
lvii, 811. 
(4) Incr, W. H.: Jour. Chem. Soc., 1895, Ixvii, 155. 
(5) Kay, H.D., anp Rapsr, H.S.: Biochem. Jour., 1924, xviii, 153. 
(6) Nrusere, C., AND RincsEr, M.: Biochem. Z., 1915, lxxi, 226. 
(7) Paourini, V.: Gazz. chim. ital., 1902, xxxii, I, 405. 
(8) Puscu, M.: Arch. d. Pharmazie, 1894, cexxxii, 186. 
(9) Rinecer, A. I.: Jour. Biol. Chem., 1912, xii, 223. 
(10) Rinarr, A. I., FRanxe., E. M., anp Jonas, L.: Jour. Biol. Chem., 1913, 
xiv, 539. 
(11) Rosz, W. C.: Jour. Pharmacol. and Exper. Therap., 1924, xxiv, 123. 
(12) Rossz, W. C.: Jour. Pharmacol. and Exper. Therap., 1924, xxiv, 147. 
(13) Rosz, W. C., Wespmr, C. J.,. Cortey, R. C., anp Jackson, R. W.: Jour. 
Pharmacol. and Exper. Therap.. 1925, xxv, 59. 
(14) Rosz, W. C., anp Dimmirt, P. S.: Jour. Pharmacol. and Exper. Therap., 
1925, xxv, 65. 
(15) Satant, W., anp Swanson, A. M.: Jour. Pharmacol. and Exper. Therap., 
1918, xi, 48. 
(16) SHarrer, P. A., anpD Hartmann, A, F.: Jour. Biol. Chem., 1920-21, xlv, 365. 
(17) THorne, L. T.: Jour. Chem. Soc., 1881, xxxix, 543. 


ae ol I wm y 


> 


UAL TE YAN 


The writer was born in Tower Hill, Illinois, June 
5, 1901. He received his grammar and high school 
training in the public schools of ower Hill and 
Pana, Illinois. 

He entered the University of Illinois in 1918 and 
was graduated in 1921. He received the degree of 
Master of Arts in 1922. 

His appointments held are as follows:— 


1921-22 Scholar in chemistry. 
1922-24 Assistant in physiological chemistry. 


PUBLICATION 


Studies in Uric Acid Metabolism. Lewis, H. B., 
anid GOLle yard sDIOL Oe) lo 25 mela 3 


MONAHAN 


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